The present invention, based on the discovery of a new biological phenomena, provides methods and compositions for use in identifying agents that modulate the phosphorylation of survivin, the interation between survivin and p34cdc2cyclin B1 kinase complex, and the interaction between survivin and caspase-9. Related methods and compositions can be used to modulate survivin regulated apoptosis.
Programmed cell death (sometimes referred to as apoptosis) is distinguishable, both morphologically and functionally, from necrosis. Programmed cell death is a natural form of death that organisms use to dispose of cells. Cells dying by programmed cell death usually shrink, rarely lyse, and are efficiently removed from the organism (rapidly recognized and engulfed by macrophages) without the appearance of inflammation (Michael Hengartner, xe2x80x9cCell Death and Aging, Molecular Mechanisms of,xe2x80x9d In Molecular Biology and Biotechnology 158-62 (ed. R. A. Meyers, 1995)).
Apoptosis was initially used to describe a subset of programmed cell deaths sharing a particular set of morphological features which include membrane blebbing, shrinkage of cytoplasm, chromatic condensation and formation of a xe2x80x9cDNA ladder.xe2x80x9d During apoptosis, cells lose their cell junctions and microvilli, the cytoplasm condenses and nuclear chromatin marginates into a number of discrete masses. While the nucleus fragments, the cytoplasm contracts and mitochondria and ribosomes become densely compacted. After dilation of the endoplasmic reticulum and its fusion with the plasma membrane, the cell breaks up into several membrane bound vesicles, referred to as apoptotic bodies, which are usually phagocytosed by adjacent cells. Activation of particular genes such as tumor suppressor genes in vertebrates is thought to be necessary for apoptosis to occur. Apoptosis induced by numerous cytotoxic agents can be suppressed by expression of the gene bcl-2, which produces a cytoplasmic protein Bcl-2 (The Encyclopedia of Molecular Biology 67 (ed. John Kendrew et al., Blackwell Science; Oxford, England, 1994).
Survivin has recently been identified as a novel inhibitor of apoptosis (IAP). The gene encoding survivin is located on chromosome 17q25. Survivin is a 16.5 kD cytoplasmic protein containing a single partially conserved BIR domain, and a highly charged carboxyl-terminus coiled-coil region instead of a RING finger, which inhibits apoptosis induced by growth factor (IL-3) withdrawal when transferred in B cell precursors (Ambrosini, G. et al., 1997). Unlike other members of the IAP family, survivin has only one BIR domain and does not have a carboxy-terminal RING finger. Instead, survivin has a carboxy-terminus coiled-coil region. Based on overall sequence conservation, the absence of a carboxy terminus RING finger and the presence of a single, partially conserved, BIR domain, survivin is the most distantly related member of the IAP family, sharing the highest degree of similarity with NAIP (Roy, N. et al., 1995). Additionally, unlike other IAP proteins, survivin is undetectable in adult tissues, but becomes prominently expressed in all the most common human cancers of lung, colon, breast, pancreas, and prostate, and in xcx9c50% of high-grade non-Hodgkin""s lymphomas, in vivo.
Expression of survivin in embryonic and fetal development may contribute to tissue homeostasis and differentiation that is independent of bcl-2 (Adida et al., 1998) . Aberrations of this survivin-associated developmental pathway may result in prominent re-expression of survivin in neoplasia and abnormally prolonged cell viability (Adida et al., 1998).
Deregulation of programmed cell death has emerged as a primary mechanism contributing to the pathogenesis of various human diseases including cancer. While the impact of anti-apoptosis genes in neoplasia has focused, for example, on the role of bcl-2 in follicular lymphoma, a potential distribution of IAP proteins, such as survivin, has only begun to been investigated. Survivin is rarely present in adult tissues but has been detected in adenocarcinoma of the pancreas, breast adenocarcinoma, colon cancer, head and neck squamous cell carcinoma, neuroblastoma, malignant thymoma, prostate cancer, and benign prostate hyperplasia (see U.S. Ser. No. 08/975,080). This expression pattern suggests that overexpression of survivin or alterations in survivin gene regulation may commonly occur during tumorigenesis.
Living organisms are composed of cells, whose growth and division require a regular sequence of events and processes that comprise the cell cycle. Some cell cycle events are continuous (e.g., synthesis of proteins and lipids), whereas others are discontinuous (e.g., DNA synthesis). Two discontinuous processes for cell survival are the replication of DNA and the segregation of chromosomes to the daughters of cell division during mitosis. If either of these steps are performed inaccurately, the daughter cells will be different from each other and will almost certainly be flawed. Segregation of chromosomes occurs during mitosis, normally a relatively brief period in the cell cycle, which culminates in the highly visible act of cell division (e.g., cytokinesis). The remainder of cell cycle comprises interphase, during which growth occurs. Chromosome replication occurs in eukaryotic cells only during interphase; and replication and segregation are mutually exclusive processes.
Interphase is subdivided into the S phase when DNA synthesis occurs and the gaps separating S phase from mitosis. G1 is the gap after mitosis, before DNA synthesis starts; G2 is the gap after DNA synthesis is complete, before mitosis and cell division. Cellular constituents direct the cell cycle by acting as regulatory elements.
One of the central functions of apoptosis (programmed cell death) in maintaining homeostasis is the elimination of damaged and potentially harmful cells (Vaux and Korsmeyer, 1999). For this process to be effective, the apoptotic machinery must constantly couple to surveillance mechanisms, i.e. xe2x80x9ccheckpointsxe2x80x9d, sensing DNA damage, adverse environmental conditions, and oncogene or viral transformation (Hunter, 1997; Paulowich et al., 1997). Checkpoint activation under these conditions initiates apoptosis (Evan and Littlewood, 1998) via the assembly of an evolutionary conserved xe2x80x9capoptosomexe2x80x9d, which in mammalian cells comprises an upstream cell death protease, caspase-9, the adapter/cofactor protein Apaf-1, mitochondria-derived cytochrome c and dATP/ATP (Green, 1998). Although it is debated how apoptosome assembly promotes caspase-9 catalytic activity (Rodriguez and Lazebnik, 1999; Zou et al., 1999), this process culminates with downstream activation of effector caspases and cleavage of critical cellular substrates (Salvesen and Dixit, 1997; Thomberry and Lazebnik, 1998). A similar paradigm linking apoptosis control to checkpoint activation (Levine, 1997), has been extended to surveillance mechanisms presiding over cell cycle transitions (Pines, 1999), assembly of a bipolar mitotic apparatus (Merdes and Cleveland, 1997), preservation of ploidy (Nicklas, 1997), and timing of cytokinesis (Field et al., 1999). In this context, dysregulated expression of apoptosis inhibitors bcl-2 and bcl-XL has been shown to restrain S phase entry (Linette et al., 1996), promote cell cycle exit (Huang et al., 1997), and cause aneuploidy (Minn et al., 1996), further demonstrating a role of the apoptotic machinery in cell-cycle progression.
Survivin is expressed in G2/M in a cell cycle-dependent manner, and localized to mitotic spindle microtubules and intercellular acto-myosin bridges, i.e. midbodies, during cell division (Li et al., 1998). Interference with this topography, or blocking survivin expression, caused increased caspase-3 activity in G2/M (Li et al., 1998), and a profound dysregulation of mitotic progression (Li et al., 1999), suggesting that survivin may regulate a novel apoptotic checkpoint at cell division. This pathway was dramatically exploited in cancer (Ambrosini et al., 1997), where survivin was identified as one of the top four xe2x80x9ctranscriptomesxe2x80x9d out of 3.5 millions mRNAs, uniformly expressed in cancer, but not in normal tissues (Velculescu et al., 1999). Additionally, it has been shown that transformed cells are exquisitely sensitive to manipulation at this mitotic checkpoint as interference with survivin expression and function using dominant-negative mutants with point mutations in the conserved baculovirus IAP repeat (BIR) domain or survivin antisense resulted in aberrant mitoses (Li et al., 1999) and spontaneous apoptosis (Ambrosini et al., 1999; Grossman et al., 1999a; Grossman et al., 1999b). This phenotype is unique to survivin and not observed with other apoptosis inhibitors potentially contributing to neoplasia, as antisense inhibition of Bcl-2 increased sensitivity to apoptosis but did not in itself induce cell death (Jansen et al. 1998).
The present invention identifies a mechanism by which survivin may integrate the control of cell division with the regulation of apoptosis in mammalian cells. The present invention also provides two classes of survivin antagonists that modulate the expression of survivin and interfere with the antiapoptotic survivin pathway in melanoma tumors in vivo. Further, the present invention provides a method of inhibiting the growth of tumor comprising administering an effective amount of a survivin antagonist to the tumor.
The present invention is based in part on the finding that survivin is phosphorylated by the main mitotic kinase complex, p34cdc2-cyclin B1 (Nurse, 1994), and that this process is essential to preserve viability of cells traversing mitosis.
The present invention is also based in part on the finding that lack of survivin phosphorylation by p34cdc2 causes dissociation of the survivin-active caspase-9 complex, selective mislocalization of caspase-9 from midbodies, and caspase-9-dependent apoptosis of cells traversing mitosis.
The present invention provides a method of identifying an agent that modulates the phosphorylation of survivin comprising the steps of incubating survivin and p34cdc2-cyclin B1 kinase complex with an agent and determining whether the agent modulates the phosphorylation of survivin, thereby identifying an agent that modulates phosphorylation of survivin.
The present invention further provides methods of identifying an agent that modulates the interaction of survivin and p34cdc2-cyclin B1 kinase complex.
The invention also provides methods of modulating the interaction between survivin and p34cdc2-cyclin B1 kinase complex comprising the step of administering an effective amount of an agent which modulates at least one interaction between Survivin and p34cdc2-cyclin B1 kinase complex.
The invention further includes methods of modulating apoptosis in a cell, comprising administering to the cell an effective amount of an agent that modulates the interaction between survivin and p34cdc2-cyclin B1 kinase complex. The invention further provides agents, compositions, and peptides that modulate the interactions between Survivin and p34cdc2-cyclin B1 kinase complex.
The present invention also provides a method of identifying agents that modulate the interaction between phosphorylated survivin and caspase-9. The invention further provides agents, compositions, and peptides that modulate the interactions between phosphorylated survivin and caspase-9.
The present invention provides survivin antagonists such as the dominant negative survivin mutant (Thr34xe2x86x92Ala) and survivin antisense nucleic acid, to modulate the expression of survivin in a cell. The invention also discloses using the survivin antagonists to inhibit the growth of tumors in vivo and in vitro.